Method for preventing vascular disease

ABSTRACT

A method for orally administering vitamin preparations is described which combine vitamin B 12  (B 12 , cobalamin) and folic acid (folate), with and without pyridoxine (B 6 ), for preventing and treating elevated serum homocysteine (HC), cystathionine (CT), methylmalonic acid (MMA), or 2-methylcitric acid (2-MCA) levels. These metabolites have been shown to be indicative of B 12  and/or folic acid deficiencies. Further, it is likely that a B 6  deficiency may be present with a B 12  or folate deficiency. The method of the invention is also for use in lowering serum HC, CT, MMA, or 2-MCA in patients with or at risk for neuropsychiatric, vascular, renal or hematologic diseases. The method of the present invention eliminates the costly and time consuming steps of distinguishing between vitamin deficiencies once a deficiency is found by measurement of serum metabolite levels. The present invention is of particular benefit to the populations at risk for elevated serum metabolite levels, such as the people over the age of 65, and populations that have or are at risk for neuropsychiatric, vascular, renal and hematologic diseases.

[0001] This application is a continuation of Ser. No. 09/793,214, filedon Feb. 26, 2001, which is a continuation of Ser. No. 09/273,754 filedMar. 22, 1999, now issued as U.S. Pat. No. 6,297,224, which is acontinuation of application no. Ser. No. 09/012,955 filed Jan. 26, 1998now issued as U.S. Pat. No. 5,795,873, which is a divisional ofapplication no. Ser. No. 07/999,499, which was filed Dec. 29, 1992, nowissued as U.S. Pat. No. 5,563,126.

FIELD OF THE INVENTION

[0002] This invention relates to the field of nutrition. Specifically,the invention is comprised of new oral vitamin preparations combiningvitamin B₁₂ (B₁₂, cobalamin) and folic acid (folate), and vitamin B₁₂,folate, and pyridoxine (B₆) for use in patients with elevated serummetabolite levels of homocysteine (HC), cystathionine (CT),methylmalonic acid (MMA), or 2-methylcitric acid (2-MCA). The elevationof these metabolites has been shown to be indicative of tissuedeficiencies of B₁₂ and/or folate and/or B₆, and related to increasedrisk of neuropsychiatric, vascular, renal and hematologic diseases. Oneembodiment of the present invention uses a non-prescription formulationcomprising between 0.3-10.0 mg B₁₂ and 0.1-0.4 mg folate, with thepreferred embodiment using 2.0 mg B₁₂ and 0.4 mg folate. Anotherembodiment of the non-prescription formulation uses 0.3-10 mg B₁₂,0.1-0.4 mg folate, and 5-75 mg B₆, with the preferred embodiment using2.0 mg B₁₂, 0.4 mg folate, and 25 mg B₆. Another embodiment of thepresent invention uses a prescription strength formulation comprisingbetween 0.3-10.0 mg B₁₂ and 0.4-1.0 mg folate, with the preferredembodiment using 2 mg B₁₂ and 1.0 mg folate. In a further embodiment ofthe present invention, a prescription strength formulation is usedcomprising 0.3-10 mg B₁₂, 0.4-1.0 mg folate, and 5-75 mg B₆, with thepreferred embodiment using 2 mg B₁₂, 1.0 mg folate, and 25 mg B₆. Theformulations of the present invention eliminate the costly andtime-consuming steps of distinguishing between vitamin deficiencies oncea deficiency is found by measurement of serum metabolite levels. Thepresent invention is of particular benefit to the populations at riskfor tissue deficiencies of B₁₂, folate, and B₆, such as people over theage of 65, and populations that have or are at risk forneuropsychiatric, vascular, renal and hematologic diseases.

BACKGROUND

[0003] Vitamins B₁₂, folate, and B₆ are required cofactors in metabolicpathways involving methionine, homocysteine, cystathionine, andcysteine. B₁₂ in the form of 5′-deoxyadenosylcobalamin is an essentialcofactor in the enzymatic conversion of methylmalonylCoA to succinylCoA.The remethylation of homocysteine (HC) to methionine catalyzed bymethionine synthase requires folate (methyltetrahydrofolate) and B₁₂ inthe form of methylcobalamin. HC is condensed with serine to formcystathionine (CT) in a reaction catalyzed by cystathionine □-synthasewhich requires B₆ (pyridoxal phosphate). CT is hydrolyzed in anotherB₆-dependent reaction to cysteine and □-ketobutyrate.

[0004] It is important to diagnose and treat B₁₂, folate, and B₆deficiencies because these deficiencies can lead to life-threateninghematologic abnormalities which are completely reversible by propertreatment. B₁₂ deficiency is a multisystem disorder with extremelyvaried clinical presentation which has been thought to occur in 0.4% ofthe population, e.g., about 1 million people in the United States.Symptoms of B₁₂ deficiency include significant anemia, displayed forexample in decreased hematocrit (e.g., <25%) or hemoglobin (e.g., <8 g%), with macrocytic red blood cells (i.e., mean cell volume generallygreater than 100 fl), or neurologic symptoms of peripheral neuropathyand/or ataxia. See, for example, Babior and Bunn (1983) in Harrison'sPrinciples of Internal Medicine, (Petersdorf et al., eds.), McGraw-HillBook Co., New York; Lee and Gardner (1984) in Textbook of FamilyPractice, 3rd Ed. (Rakel, ed.), Saunders & Co., Philadelphia). Thehematological abnormalities seen are due to intracellular folatedeficiency since folate is required for a number of essential enzymaticreactions involved in DNA and RNA synthesis and since the form of folatein serum (5-methyltetrahydrofolate) must be metabolized totetrahydrofolate by the B₁₂-dependent enzyme methionine synthase beforeit can be utilized by the RNA- and DNA-related enzymes. While it hasbeen well recognized that individuals with B₁₂ deficiency could displayneurologic disorders in the absence of anemia, such situations werebelieved to be exceptional and rare. See, Beck (1985) in Cecil Textbookof Medicine, 17th Ed., (Wyngaarden and Smith, eds.), W. B. Saunders,Philadelphia, pp. 893-900; Babior and Bunn (1987) in Harrison'sPrinciples of Internal Medicine, 11th Ed., (Braunwald et al., eds.)McGraw-Hill, New York, pp. 1498-1504; Walton (1985) in Brain's Diseasesof the Nervous System, 9th Ed., Oxford University Press, Oxford, UK. Theneurologic symptoms of B₁₂ deficiency were considered to be latemanifestations of the disease most typically occurring after the onsetof anemia or, if they occurred first, were soon to be followed by theonset of anemia. See, Woltmann (1919) Am. J. Med. Sci. 157:400-409Victor and Lear (1956) Am. J. Med. 20:896-911.

[0005] However, it has recently been shown that the textbook descriptionof severe megaloblastic anemia and combined systems disease of thenervous system is the rarest presentation of B₁₂ deficiency at thepresent time (Stabler et al. (1990) Blood 76:871-881; Carmel (1988)Arch. Int. Med. 148:1712-1714 Allen (1991) in Cecil Textbook ofMedicine, 19th Ed., (Wyngaarden and Smith, et al. eds.), W. B. Saunders,Philadelphia, pp. 846-854.). Therefore, contrary to previous teachings,patients that may benefit from B₁₂ therapy may have minimal to nohematologic changes while manifesting a wide variety of neurologic andpsychiatric abnormalities (Lindenbaum et al. (1988) N. Engl. J. Med.318:1720-1728; Greenfield and O'Flynn (1933) Lancet 2:62-63). This isparticularly true for populations at risk for B₁₂ deficiency, such asthe elderly population (Pennypacker et al. (1992) J. Am. Geriatric Soc.40: (in press).

[0006] The incidence of folate deficiency in the population is unknown,but has been thought to occur commonly in individuals with variousdegrees of alcoholism. The hematologic abnormalities seen with folatedeficiency, such as macrocytic anemia, are indistinguishable from thoseseen with B₁₂ deficiency. Folate is required for a number of essentialenzymatic reactions involved in DNA and RNA synthesis, and isparticularly important in rapidly dividing cells like those in the bonemarrow.

[0007] B₆ is required for the first step in heme synthesis and serves amajor role in transamination reactions of amino acid metabolism, indecarboxylations, and in the synthesis of the neuroactive amineshistamine, tyramine, serotonin, and □-aminobutyric acid (GABA). Clinicalmanifestations include microcytic hypochromic anemia, characteristicskin changes of dermatitis and acrodynia, muscular weakness, and avariety of neuropsychiatric abnormalities including hyperirritability,epileptiform convulsions, depression and confusion (Newberne and Conner(1989) in Clinical Biochemistry of Domestic Animals, Academic Press, SanDiego, pp. 796-834).

[0008] Vitamin deficiencies are generally determined by measurement ofserum levels. Normal serum B₁₂ levels are 200-900 pg/ml, with levels ofless than 100 pg/ml being said to indicate clinically significantdeficiency (Beck (1985) supra) However, serum B₁₂ levels are arelatively insensitive determinant of B₁₂ deficiency in that only 50% ofpatients with clinically confirmed B₁₂ deficiency have levels less than100 pg/ml, 40% are 100-200 pg/ml, and at least 5-10% have values in the200-300 pg/ml range. Diagnosis is further complicated by the fact that2.5% of normal subjects (6,250,000 people in the U.S.) have low serumB₁₂ levels (Allen (1991) supra), with no evidence of B₁₂ deficiency andare unlikely to benefit from B₁₂ therapy (Schilling et al. (1983) Clin.Chem. 29:582; Stabler (1990) supra).

[0009] Normal serum folate levels are 2.5-20 ng/ml, with levels lessthan 2.5 ng/ml indicating the possibility of clinically significantdeficiency. Like B₁₂ serum levels, however, serum folate levels are arelatively insensitive measure in that only 50-75% of patients withfolate deficiency have levels less than 2.5% ng/ml, with most of theremaining 25-50% being in the 2.5-5.0 ng/ml range (Allen (1991) in CecilTextbook of Medicine, 19th Ed., su ra) The development of sensitiveserum metabolite assays for HC, CT, MMA, and 2-MCA has allowed therelationship between metabolite levels and vitamin deficiencies to beinvestigated (Stabler et al. (1987) Anal. Biochem. 162:185-196; Stableret al. (1986) J. Clin. Invest. 77:1606-1612; Stabler et al. (1988) J.Clin. Invest. 81:466-474). It has been found that elevated serum levelsof HC and MMA are clinically useful tests of functional intracellulardeficiencies of B₁₂ and folate, with elevated HC levels seen with bothB₁₂ and folate deficiencies, and elevated MMA levels seen with a B₁₂deficiency (Allen et al. (1990) Am. J. Hematol. 34:90-98 Lindenbaum etal. (1990) Am. J. Hematol. 34:99-107; Lindenbaum et al. (1988) N. Engl.J. Med. 318:1720-1728; Beck (1991) in Neuropsychiatric Consequences ofCobalamin Deficiency, Mosby Year Book 36:33-56 Moelby et al. (1990)228:373-378; Ueland and Refsum (19890 J. Lab. Clin. Med. 114:473-501;Pennypacker et al. (1992) supra). Increased serum levels of CT are seenin both deficiencies and 2-MCA is elevated in B₁₂ deficiency (Allen etal. (1991) in Proceedings of the 1 st International Congress on Vitaminsand Biofactors in Life Science, Kobe (Japan) ; Allen et al. (1993)Metabolism (in press)). HC and CT may be elevated in patients withintracellular deficiency of B₆, but this has not been as well documented(Park and Linkswiler (1970) J. Nutr. 100:110-116; Smolin and Benvange(1982) J. Nutr. 112:1264-1272).

[0010] Elevated serum metabolite levels are observed in disease statesother than classic vitamin deficiencies. For example, elevated HC levelshave been observed in the presence of vascular disease. The homocysteinetheory of atherosclerosis, formulated by McCully and Wilson (1975)Atherosclerosis 22:215-227, suggests that high levels of HC areresponsible for the vascular lesions seen in homocystinuria, a geneticdefect caused by a deficiency in the enzyme cystathionine □-synthase.The theory also implies that moderate elevations of HC might beassociated with increased risk for vascular disease (Ueland et al.(1992) in Atherosclerotic Cardiovascular Disease, Hemostasis, andEndothelial Function (Francis, Jr., ed.), Marcel Dekker, Inc., New York,pp. 183-236). Moderate hyperhomocysteinemia has been shown to befrequently present in cases of stroke and to be independent of otherstroke risk factors (Brattstrom et al. (1992) Eur. J. Clin. Invest.22:214-221). Clinical and experimental evidence demonstrates thatpatients who are homozygotes for cystathionine □-synthase deficiencyhave a markedly increased incidence of vascular disease and thrombosis.A number of studies (see, Clarke et al. (1991) N. Engl. J. Med.324:1149-1155) strongly suggest that heterozygotes for a deficiency ofcystathionine β-synthase also have an increased incidence of vasculardisease and thrombosis and that such heterozygotes may constitute asmany as one-third of all patients who develop strokes, heart attacks, orperipheral vascular disease under age 50. It is also likely that suchheterozygotes are also at increased risk for vascular disease andthrombosis after age 50. Since the incidence of heterozygosity forcystathionine β-synthase deficiency is estimated to be 1 in 60-70, thismeans that there are approximately 4 million heterozygotes in the U.S.It is also possible that patients with vascular disease due to othercauses, such as hypercholesterolemia, would also benefit from a decreasein their serum HC levels even if their existing levels are only slightlyelevated or actually within the normal range.

[0011] Renal disease is another condition that gives rise to elevatedlevels of serum metabolites. Approximately 75% of patients with renaldisease have elevated serum concentrations of HC, CT, MMA, and 2-MCA.Since patients with renal disease have a significant incidence andmarked acceleration of vascular disease, it might be beneficial to lowertheir serum metabolite levels, especially that of HC.

[0012] An increasing prevalence of low serum B₁₂ concentrations withadvancing age has been found by many but not all investigators (Baileyet al. (1980) J. Am. Geriatr. Soc. 28:276-278 Eisborg et al. (1976) ActaMed. Scand. 200:309-314; Niisson-Ehle et al. (1989) Dig. Dis. Sci.34:716-723; Norman (1985) 33:374; Hitzhusen et al. (1986) Am. J. Clin.Pathol. 85:3236), folate (Magnus et al. (1982) Scan. J. Haematol.28:360-366; Blundell et al. (1985) J. Clin. Pathol. 38:1179-1184 Elwoodet al. (1971) Br. J. Haematol. 21:557-563; Garryet al. (1984) J. Am.Geriatr. Soc. 32:71926; Hanger et al. (1991) J. Am. Geriatr. Soc.39:1155-1159), and B₆ (Ranke et al. (1960) J. Gerontol. 15:41-44; Roseet al. (1976) Am. J. Clin. Nutr. 29:847-853; Baker et al. (1979) J. Am.Geriatr. Soc. 27:444-450). Moreover, prevalence estimates for thesevitamin deficiencies vary widely depending on the population groupsstudied. It has been unclear whether this increased prevalence is anormal age related phenomena or a true reflection of tissue vitamindeficiency and whether the low serum vitamin concentrations are areliable indicator of functional intracellular deficiency.

[0013] It is difficult, expensive and time-consuming to distinguishbetween deficiencies of vitamins B₁₂, folate, and B₆. The hematologicabnormalities seen with B₁₂ deficiency are indistinguishable from thoseseen with folate deficiency. Similarly to a B₁₂ deficiency, B₆deficiencies also result in hematologic as well as neuropsychiatricabnormalities. The traditional methods of determining deficiencies bymeasurement of serum vitamin levels are often insensitive. As a result,in order to determine if and which vitamin deficiency is present, apatient will be treated with one vitamin at a time and the response tothat vitamin determined by normalization of serum vitamin levels and thecorrection of hematologic abnormalities. These steps are then repeatedwith each vitamin. This method of treatment is both expensive andtime-consuming. In the presence of multiple deficiencies, the diagnosisof vitamin deficiencies is further confused and give rise to thedangerous possibility that only one deficiency will be treated. Forexample, the hematologic abnormalities seen with a B₁₂ deficiency willrespond to treatment with folate alone. However, the neuropsychiatricabnormalities caused by the B₁₂ deficiency will not be corrected and mayindeed by worsened.

[0014] It has now been discovered for the first time that the prevalenceof intracellular deficiencies of vitamins B₁₂, folate, and B₆, alone orin combination, is substantially higher than that previously estimatedby measurement of serum vitamin concentrations. The present disclosureestablishes that tissue deficiencies of one or more of the vitamins B₁₂,folate and B₆, as demonstrated by the elevated metaboliteconcentrations, occurs commonly in the elderly population even whenserum vitamin levels are normal. Based on this new discovery, thepresent invention addresses the problem of distinguishing betweenvitamin deficiencies when low, low-normal, or normal serum vitaminconcentrations are found by providing formulations for the treatment ofhigh serum metabolites and at-risk populations for combinations of oneor more tissue deficiencies of vitamins B₁₂, folate, and B₆.

[0015] Hathcock and Troendle (1991) JAMA 265:96-97, have suggested thetreatment of pernicious anemia with an oral pill containing 300 to 1000ug or more per day of B₁₂. However, contrary to the present invention,Hathcock and Troendle teach away from combining B₁₂ therapy with folate,since “if the oral cobalamin therapy should fail to maintain adequatelevels, folate might provide protection against development of anemiawhile permitting nerve damage from cobalamin deficiency.”

[0016] U.S. Pat. No. 4,945,083, issued Jul. 31, 1990 to Jansen,entitled: Safe Oral Folic-Acid-Containing Vitamin Preparation, describesa oral vitamin preparation comprising 0.1-1.0 mg B₁₂ and 0.1-1.0 mgfolate for the treatment or prevention of megaloblastic anemia. Thisformulation presents a problem in the case of a B₁₂ deficient patient,in that the 0.5 mg folate may correct the hematologic abnormalitiespresent, but the 0.5 mg B₁₂ dose may be insufficient to correct a B₁₂deficiency due to inadequate intrinsic factor. By contrast, theformulation of the present invention teaches the use of the combinationof B₁₂ and folate, and of B₁₂, folate and B₆, sufficient to treat eithersingle or multiple deficiencies of B₁₂, folate, and B₆. The presentinvention does not rely on the determination of vitamin deficiencies bythe measurement of serum vitamin levels, but uses the more sensitivemeasurement of elevated serum metabolites of HC, CT, MMA, and 2-MCA,shown to be related to the presence of B ₁₂ and/or folate and/or to B6deficiencies or to the presence of the increased risk ofneuropsychiatric, vascular, renal, and hematologic diseases.

[0017] It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention as claimed.

SUMMARY OF THE INVENTION

[0018] This invention includes a method for orally administering two newvitamin preparations containing vitamin B₁₂ and folate, and vitamin B₁₂,folate and B₆, for the treatment of patients with elevated serummetabolites, such as homocysteine, cystathionine, methylmalonic acid,and 2-methylcitric acid, as well as populations at risk for tissuedeficiencies in one or more of the vitamins B₁₂, folate, and B₆ or forneuropsychiatric, vascular, renal, or hematologic diseases.

[0019] One embodiment of the present invention uses an over-the-counterformulation comprised of between 0.3-10 mg CN-cobalamin (B₁₂) and0.1-0.4 mg folate. Another embodiment of the non-prescriptionformulation uses 0.3-10 mg B₁₂, 0.1-0.4 mg folate, and 5-75 mg B₆.Preferred embodiments of the over-the-counter formulation are comprisedof about 2.0 mg B₁₂ and 0.4 mg folate, and 2.0 mgB₁₂, 0.4 mg folate, and25 mg B₆, respectively.

[0020] Another embodiment of the present invention uses a prescriptionformulation comprised of between 0.3-10 mg CN-cobalamin (B₁₂) and0.4-10.0 mg folate. Another embodiment of the prescription formulationof the present invention uses 0.3-10 mg B₁₂, 0.4-10.0 mg folate, and5-75 mg B₆. Preferred embodiments of the prescription formulation useabout 2.0 mg B₁₂ and 1.0 mg folate, and 2.0 mg B₁₂, 1.0 mg folate, and25 mg B₆, respectively.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 shows the distribution of serum B₁₂ levels for a populationof elderly outpatients (ages 65-99, n=152) and a normal population (ages17-65, n=100).

[0022]FIG. 2 shows serum MMA levels for a population of elderlyoutpatients with serum B₁₂ values <300 pg/ml (ages 65-99, n=38/152) anda normal population with serum B₁₂ values <300 pg/ml (ages 17-65,n=10/100).

[0023]FIG. 3 shows serum HC levels for a population of elderlyoutpatients with serum B₁₂ values <300 pg/ml (ages 65-99, n=38/152) anda normal population with serum B,₂ values <300 pg/ml (ages 17-65,n=10/100).

[0024]FIG. 4 shows serum MMA levels before and after treatment withparenteral cobalamin for a population of elderly outpatients withelevated MMA values and serum B₁₂ values <300 pg/ml (ages 65-99,n=15/38).

[0025]FIG. 5 shows serum HC levels before and after treatment withparenteral cobalamin for a population of elderly outpatients withelevated HC values and serum B₁₂ values of <300 pg/ml (ages 65-99,n=10/38).

[0026]FIG. 6 shows the distribution of serum B₁₂ levels for a populationof elderly nursing home patients (ages 55-107, n=212) and a normalpopulation (ages 17-65, n=100).

[0027]FIG. 7 shows serum MMA levels for a population of elderly nursinghome patients with serum B₁₂ values <300 pg/ml (ages 55-107, n=29/212)and a normal population with serum B₁₂ values (ages 17-65, n=10/100).

[0028]FIG. 8 shows serum HC levels for a population of elderly nursinghome patients with serum B₁₂ values <300 pg/ml (ages 55-107, n=29/212)and a normal population with serum B12 values <300 pg/ml (ages 17-65,n=10/100).

[0029]FIG. 9 shows serum MMA levels before and after treatment withparenteral cobalamin for a population of elderly nursing home patientswith serum B₁₂ values <300 pg/ml (ages 55-107, n=14/29).

[0030]FIG. 10 shows serum HC levels before and after treatment withparenteral cobalamin for a population of elderly nursing home patientswith serum B₁₂ values <300 pg/ml (ages 55-107, n=14/29).

[0031]FIG. 11 shows the distribution of serum B₁₂ levels for apopulation of elderly patients (ages 65-99, n=548) and a normalpopulation (ages 22-63, n=1 17) (Framingham study).

DETAILED DESCRIPTION OF THE INVENTION

[0032] Reference will now be made in detail to the presently preferredembodiments of the invention, which, together with the followingexamples, serve to explain the principles of the invention.

[0033] This invention uses new oral vitamin formulations combiningvitamin B₁₂ (B₁₂, cobalamin) and folic acid (folate), and vitamin B₁₂,folate and pyridoxine (B₆). The formulations of the present inventionare for use in the treatment of elevated serum levels of one or more ofthe metabolites homocysteine (HC), cystathionine (CT), methylmalonicacid (MMA), or 2-methylcitric acid (2-MCA). The use of the formulationsof the present invention further include as a method of lowering serummetabolite levels of one or more of HC, CT, MMA, or 2-MCA, where thesemetabolite levels are not elevated but the patients are at risk for orhave neuropsychiatric, vascular, renal, or hematologic diseases.

[0034] One embodiment of the present invention uses a non-prescriptionformulation comprised of between about 0.3-10 mg CN-cobalamin (B₁₂) and0.1-0.4 mg folate. Another embodiment of the present invention uses anon-prescription formulation comprised of between about 0.3-10 mg B₁₂,0.1-0.4 mg folate, and 5-75 mg B₆. Preferred embodiments of thenon-prescription formulation are comprised of about 2.0 mg B₁₂ and 0.4mg folate, and 2.0 mg B₁₂, 0.4 mg folate, and 25 mg B₆, respectively.

[0035] Another embodiment of the present invention is comprised of aprescription formulation comprised of between about 0.3-10 mg B₁₂ and0.4-10.0 mg folate, with the preferred embodiment comprised of about 2.0mg B₁₂ and 1.0 mg folate. Another embodiment of the prescriptionstrength formulation is comprised of about 0.3-10 mg B₁₂, 0.4-10.0 mgfolate, and 5-75 mg B₆, with a preferred embodiment comprised of about2.0 mg B₁₂, 1.0 mg folate, and 25 mg B₆.

[0036] The formulations of the present invention are for the treatmentand prevention of elevated metabolite levels in at risk populations,such as the elderly, and people that have or are at risk forneuropsychiatric, vascular, renal and hematologic diseases. The presentinvention eliminates the costly and time consuming need to differentiatebetween B₁₂, folate, and B₆ deficiencies.

[0037] The administration of a daily dose of the vitamin formulations ofthe present invention provides better long-term normalization of serumHC and other metabolites than prior art formulations, and eliminates thedifficulty in differentiating between deficiencies of two or three ofthe vitamins, the difficulty in diagnosing multiple deficiencies of twoor three of the vitamins, and the expense of doing so. Further, theadministration of an oral preparation of B₁₂ and folate, with or withoutB₆, is preferred over intramuscular injections for patient convenienceand ease of administration.

[0038] For example, the inclusion of B₁₂ will be useful as a safeguardfor patients misdiagnosed as folate deficient, even though they areactually B₁₂ deficient, since treatment with folate alone in suchpatients is extremely dangerous. The danger arises from the fact thattreating a B₁₂ deficient patient with folate alone may reverse orprevent the hematologic abnormalities seen in B₁₂ deficiency, but willnot correct the neuropsychiatric abnormalities of a B₁₂ deficiency andmay actually precipitate them. Even in the absence of intrinsic factor,approximately 1% of a 2.0 mg oral dose of B₁₂ is absorbed by diffusion.Thus, approximately 20 ug of B₁₂ would be absorbed from the formulationsof the present invention which would be more than adequate even inpatients with pernicious anemia who have lost their intrinsicfactor-facilitated absorption mechanism for B₁₂. The inclusion of folatewill be of benefit since B₁₂ deficiency causes a secondary intracellulardeficiency of folate. The inclusion of folate and B₆ will also be ofbenefit in patients with mixed vitamin deficiencies.

[0039] The formulations of the present invention may be administered asa non-injectable implant or orally. Non-injectable use may be as apatch. Formulations for oral administration are preferably encapsulated.Preferably, the capsule is designed so that the formulation is releasedgastrically where bioavailability is maximized. Additional excipientsmay be included to facilitate absorption of the vitamin formulations.Diluents, flavorings, low melting point waxes, vegetable oils,lubricants, suspending agents, tablet disintegrating agents, and bindersmay also be employed.

[0040] Example 1 describes the methods used to measure serum vitamin andmetabolite levels. Example 2 describes a new study conducted with 412subjects over the age of 65 with a variety of medical conditionscorrelating the incidence of low serum vitamin levels with elevatedserum metabolite levels. A study determining the incidence of undetectedB12 deficiency and response of serum MMA and HC to B₁₂ treatment in ageriatric outpatient population is described in Example 3. Example 4describes a similar study conducted with a geriatric nursing homepopulation, and Example 5 describes a similar study conducted withanother geriatric population.

EXAMPLE 1

[0041] Methods for Measurement of Serum Vitamin and Metabolite Levels.

[0042] Serum vitamin assays. Serum vitamins B12 and folate were measuredby a quantitative radioassay method using purified intrinsic factor andpurified folate binding protein. Vitamin B6 was measured by aradioenzymatic assay method wherein serum is incubated with apoenzymetyrosine-decarboxylase, C₁₄ labelled tyrosine is added to start theenzymatic reaction which is stopped with HCl. Subsequently the freeC₁₄-labelled CO₂ is adsorbed by a KOH impregnated filtering paper. Themeasured C₁₄ activity is directly proportional to the B₆ (pyridoxalphosphate) concentration (Laboratory Bioscientia, Germany).

[0043] Serum metabolite assays. Serum metabolite assays for homocysteineand methylmalonic acid were conducted by the capillary gaschromatography and mass spectrometry methods of Marcell et al. (1985)Anal. Biochem. 150:58; Stabler et al. (1987) supra, and Allen et al.(1990) Am. J. Hematol. 34:90-98. Serum cystathionine levels were assayedby the method of Stabler et al. (1992) Blood (submitted). Serum2-methylcitric acid was assayed by the method of Allen et al. (1993)Metabolism supra.

[0044] Statistical methods. Statistical analysis was done with the SASstatistical package (version 6.06). Nonparametric data for two or moregroups were tested with the two sample Wilcoxon rank sum test (withBonferroni's correction for the significance level ax) and the KruskallWallis test. From the results of the healthy young subjects referenceintervals were calculated. Since the frequency distribution of thevalues of each parameter were markedly abnormal they were transformed tonormal distributions using log transformation. The sample prevalence pwith 95% confidence intervals of low serum vitamins B₁₂, folate, and B₆concentrations was calculated as (p±2p (1−p)/n×100 wherein n is thetotal sample size, p is the number of low serum vitaminconcentrations/n; low serum concentrations are defined as <mean −2 S.D.

EXAMPLE 2

[0045] Incidence of Elevated MMA, 2-MCA, HC, and CT Levels in theGeriatric Population.

[0046] The serum concentrations of B₁₂, folate, and B₆ were measured in412 subjects over the age of 65 (subgroups A-D), and in 99 healthycontrol subjects aged 20-55 years (subgroup E). The geriatric subgroupswere defined as follows: A, 110 patients with atherosclerosis; B, 98patients with neuropsychiatric disorders; C, 102 patients withatherosclerosis and multiple diseases including rheumatoid arthritis anddiabetes; D, 102 subjects who were healthy.

[0047] Venous blood was obtained from all subjects in the morning afteran overnight fast. The blood was spun within one hour after collectionand the serum was transported in dry ice to the central laboratory.Serum vitamins B₁₂ and folate were measured as described in Example 1with a vitamin B₁₂/folate dual RIA kit (CT301/CT302 Amersham Buchier,UK). Vitamin B₆ and serum metabolites were measured as described inExample 1.

[0048] Since renal function can influence serum metaboliteconcentrations (Ueland and Refsum (1989) supra Moelby et al. (1992)Scand. J. Clin. Lab. Invest. 52:351-354), serum creatinineconcentrations were measured in all subjects by the Jaffe photometricmethod (Laboratory Bioscientia, Germany). Normal range was 62-124μmol/L. Creatinine clearance was calculated using the formulation ofCockroft and Gault (1976) Nephron 16:31-41.

[0049] Normal ranges for serum vitamin and metabolite levels weredetermined by the mean ±2 standard deviations after log normalizationusing the values from subgroup E. Results are shown in Table 1: TABLE 1INCIDENCE OF LOW SERUM VITAMN AND HIGH METABOLITE LEVELS IN GERIATRICPOPULATIONS A-D AND A YOUNGER HEALTHY POPULATION E. Folic Group B₁₂ AcidB₆ MMA 2-MCA HC CT A 6% 12% 48% 36% 44% 55% 64% B 6% 19% 53% 47% 39% 59% 6% C 3% 10% 50% 32% 45% 39% 73% D 6%  6% 17% 26% 23% 38% 41% E 2%  1% 1%  3%  6%  2%  4%

[0050] There was a rough correlation with low vitamin levels andelevated metabolites, but many of the patients with elevated metaboliteshad low normal or normal vitamin levels. Correlations between clinicalabnormalities within groups A, B, and C were not present. Patients weretreated with weekly injections of a multi-vitamin preparation containing1.0 mg B₁₂, 1.1 mg folate, and 5 mg B₆, resulting in a marked loweringor normalization of elevated metabolite levels in virtually everyelderly patient.

[0051] These data support the conclusions that there is an increasedincidence of low levels of serum B₁₂, folate, and B₆ in the geriatricpopulation, and that serum MMA, 2-MCA, HC and CT are elevated in an evenhigher percentage of geriatric patients. The presence of elevated levelsof one or more of the metabolites HC, CT, MMA, or 2-MCA indicate atissue or intracellular deficiency of one or more of the vitamins B₁₂,folate and B₆. It not possible to tell without expensive,time-consuming, and extensive testing which one vitamin or pair ofvitamins, or whether all three vitamins are deficient. Theseobservations, together with the fact that elevated metabolite levels arecorrected by parenteral therapy with a combination of vitamins B₁₂,folate, and B₆, indicate that a tissue deficiency of one or more ofthese vitamins occurs commonly in the geriatric population and thatmeasurement of serum vitamin levels alone is an inadequate method foridentifying such deficiencies.

EXAMPLE 3

[0052] Determination of Serum B₁₂ Folate, MMA, HC, CT and 2-MCA Levelsin a Geriatric Outpatient Population.

[0053] A study was conducted with 152 elderly outpatient subjects tomeasure the prevalence of B₁₂ deficiency in geriatric outpatients asdetermined by both low serum B₁₂ levels and elevations of MMA and HC,and to determine the response to B₁₂ treatment. Blood samples wereobtained on 152 consecutive geriatric outpatients, ages 65-99. Controlvalues were determined from 100 subjects, ages 17-65. Serum B₁₂ folate,MMA, HC, CT, and 2-MCA levels were obtained for each patient, shown inTable 2. The significance of the results marked as “**” in Table 2 areas follows: B₁₂ levels of <200 pg/ml; folate <3.8 ng/ml;homocysteine >16.2 uM; MMA >271 nM; CT >342 nM; and 2-MCA >228 nM. SerumMMA, HC, CT, and 2-MCA levels were measured as described in Example 1.Serum B₁₂ and folate were measured as described in Example 1 using aComing humophase kit (CIBA-Corning, Medfield, Mass.) with the normalrange defined as 200-800 pg/ml for B₁₂ and 3.8 ng/ml for folate. Afterevaluation, patients received weekly parenteral cyanocobalamininjections (1,000 ug IM) for 8 weeks, followed by monthly injections.Repeat laboratory and clinical assessments were administered at 8 weeksand at 6 months.

[0054] Results show that 25% of the subjects had a serum B₁₂ level <300pg/ml and 8.5% had a low level of <200 pg/ml. FIG. 1 shows the shiftseen in elderly subject towards lower serum B₁₂ levels. More than halfof the subjects with low or low-normal serum B₁₂ levels had elevationsof MMA (FIG. 2) and/or HC (FIG. 3) greater than 3 S.D. above the meansin normals and representing 14.5% of the total screened population.

[0055] Patients with low and low/normal serum B₁₂ levels were treatedwith weekly injections of 1.0 mg B₁₂. Parenteral B₁₂ administrationcaused elevated metabolite levels to fall to or towards normal (FIGS. 4and 5) in every subject treated with B₁₂. It appears that the trueprevalence of previously unrecognized B₁₂ deficiency in this elderlypopulation was at least 14.5%.

[0056] It can be seen from the data presented in Table 2 that serum B₁₂levels are insensitive for screening B₁₂ deficiencies since similarnumbers of patients with low normal serum B₁₂ levels of 201-300 pg/mlcompared with patients with low B₁₂ levels (<200 pg/ml) had markedlyelevated metabolites which fell with B₁₂ treatment. Further, this studyshows that elderly patients have a high incidence (at least 14.5%) ofunrecognized B₁₂ deficiency, detectable by measurement of serum HC andMMA levels in patients with serum B₁₂ levels <300 pg/ml.

[0057] A further finding in this study emphasizes the need to treatelevated metabolite levels with a combination of vitamin B₁₂ and folatewith or without B₆. Some of the patients exhibiting elevated metabolitelevels did not fully respond to B₁₂ treatment. This may indicate aconcomitant deficiency of folate and/or B₆. The lack of response to B₁₂treatment could result from a deficiency of one, a pair, or all threevitamins. However, it would be expensive and time-consuming to attemptto distinguish between the vitamin deficiencies.

[0058] Another, and perhaps the most important, finding in this study isthe large number of patients with serum B₁₂>300 pg/ml who have elevatedvalues for one or more metabolites as indicated by a “**” next to theindividual values. As can readily be seen in Table 2, there are manyexamples of elevated value for MMA and/or 2-MCA at all levels of serumB₁₂ including the mid-normal (300-600 pg/ml), the high-normal (600-800pg/ml), and even the elevated (>800 pg/ml) ranges. The same is true forelevations of HC and CT. In some patients the serum folate is low,indicating that folate deficiency may be present, but in many cases bothB₁₂ and folate levels are normal. B₆ levels were not performed in thisstudy, but B₆ deficiency would not be expected to cause elevations ofMMA or 2-MCA. Thus in many patients it is not clear which vitamin, orpair of vitamins, or whether all three vitamins is or are deficient. Onecould pick a single vitamin, often at random, with which to treat apatient for several weeks or months, and then repeat measurement ofmetabolite levels to determine if a partial or full correction hadoccurred. If there was no response, one could try another vitamin, or ifthere was a partial response one could add a second vitamin, and thenrepeat metabolite measurement after several weeks or months. If therewas still no response, one could try the third vitamin, or if there wasa partial response, one could try a different pair of vitamins.Eventually one could determine whether an individual vitamin, aparticular pair of vitamins, or all three vitamins were required tonormalize or maximally reduce the metabolite levels, but it would oftenrequire months or even a year to make this determination. Such adetermination would be expensive. In addition, a patient who wasoptimally treated with a single vitamin or pair of vitamins mightsubsequently develop a deficiency of one or even two of the othervitamins as evidenced by a re-elevation or increase in the levels of oneor more metabolites. Therapeutic testing could be reinitiated andcontinued as described above, although this would also be time-consumingand expensive.

[0059] It requires less time and expense to treat patients with elevatedmetabolite levels with a combination of vitamin B₁₂ and folate, or acombination of vitamin B₁₂, folate and vitamin B₆. The utility of theapproach of the present invention is appreciated only after it istaught, for the first time in the present disclosure, that a deficiencyof one or more of the three vitamins occurs commonly in the elderlypopulation as evidenced by elevation of one or more metabolites, i.e.,MMA, 2-MCA, HC and CT.

EXAMPLE 4

[0060] Determination of Serum B₁₂, Folate, MMA, and HC Levels in aGeriatric Nursing Home Population.

[0061] A study was conducted with 212 elderly nursing home patients todetermine serum B₁₂, folate, MMA, and HC levels (Table 3). Thesignificance of the results shown in Table 3 marked with “**” are asdescribed for Table 2 (Example 3). The control group consisted of 100subjects between the ages of 17-65 years. As in the study described inExample 3, the elderly population exhibited a shift to lower serum B₁₂levels (FIG. 6), elevated serum MMA (FIG. 7) and HC (FIG. 8) levels.Parenteral administration of B₁₂ 1 mg per week for 8 weeks to those withserum B₁₂<300 pg/ml caused elevated MMA (FIG. 9) and HC (FIG. 10) levelsto fall to or towards normal.

[0062] As in the study reported in Example 3, a further finding in thisstudy emphasizes the need to treat elevated metabolite levels with acombination of vitamins B₁₂ and folate, with or without B₆. Some of thepatients exhibiting elevated metabolite levels did not fully respond toB₁₂ treatment. This may indicate a concomitant deficiency of folateand/or B₆. The lack of response to B₁₂ treatment could result from adeficiency of one, a pair, or all three vitamins. However, it would beexpensive and time-consuming to attempt to distinguish between thevitamin deficiencies.

[0063] Again, an important finding in this study is the large number ofpatients with serum B₁₂>300 pg/ml who have elevated values for one ormore metabolites as indicated by a “**” next to the individual values.As is seen in Table 3, there are many examples of elevated values forMMA at all levels of serum B₁₂ including the mid-normal (300-600 pg/ml),the high-normal (600-800 pg/ml), and even the elevated (>800 pg/ml)ranges. The same is true for elevations of HC. In some patients theserum folate is low, indicating that folate deficiency may be present,but in many cases both B₁₂ and folate levels are normal. B₆ levels werenot performed in this study, but B₆ deficiency would not be expected tocause elevations of MMA. Thus, again it is not clear which vitamin, orpair of vitamins, or whether all three vitamins is or are deficient. Onecould pick a single vitamin with which to treat a patient for severalweeks or months, and then repeat measurement of metabolite levels todetermine if a partial or full correction had occurred. If there was noresponse, one could try another vitamin, or if there was a partialresponse one could add a second vitamin, and then repeat metabolitemeasurement after several weeks or months. If there was still noresponse, one could try the third vitamin, or if there was a partialresponse, one could try a different pair of vitamins. Eventually onecould determine whether an individual vitamin, a particular pair ofvitamins, or all three vitamins were required to normalize or maximallyreduce the metabolite levels, but it would often require months or evena year to make this determination. Such a determination would beexpensive. In addition, a patient who was optimally treated with asingle vitamin or pair of vitamins might subsequently develop adeficiency of one or even two of the other vitamins as evidenced by are-elevation or increase in the levels of one or more metabolites.Therapeutic testing could be reinitiated and continued as describedabove, although this would also be time-consuming and expensive.

[0064] It requires less time and expense to treat patients with elevatedmetabolite levels with a combination of vitamin B₁₂ and folate, or acombination of vitamin B_(12,) folate and vitamin B₆. The utility of theapproach of the present invention is appreciated only after it istaught, for the first time in the present disclosure, that a deficiencyof one or more of the three vitamins occurs commonly in the elderlypopulation as evidenced by elevation of one or more metabolites, i.e.,MMA, 2-MCA, HC and CT.

EXAMPLE 5

[0065] Determination of Serum B₁₂ Folate, I4MA, and HC Levels in aGeriatric Population.

[0066] A study was conducted with 548 elderly subjects from theFramingham study between the ages of 65-99 to determine serum B₁₂,folate, MMA, and HC levels (Table 4). The significance of the resultsshown in Table 4 (marked with “**”) are as described for Table 2(Example 2).

[0067] As in the study described in Examples 3 and 4, the elderlypopulation exhibited a shift to lower serum B₁₂ levels (FIG. 11), andelevated serum MMA and HC levels. The elderly population also exhibiteda high incidence (9.5%) of low serum folate levels (Table 4). As in thestudies reported in Examples 2, 3 and 4, the incidence of tissue orintracellular vitamin deficiencies based on elevated metabolite levelswas higher than that predicted from measurement of serum vitamin levels.

[0068] As in Examples 3 and 4 above, these results confirm theimportance of the finding that there are a large number of patients withserum B₁₂>300 pg/ml who have elevated values for one or more metabolitesas indicated by a “**” next to the individual values. As is seen inTable 4, there are many examples of elevated MMA values at all levels ofserum B₁₂ including the mid-normal (300-600 pg/ml), the high-normal(600-800 pg/ml), and even the elevated (>800 pg/ml) ranges. The same istrue for elevations of HC. In some patients the serum folate is low,indicating that folate deficiency may be present, but in many cases bothB₁₂ and folate levels are normal. B₆ levels were not performed in thisstudy, but B₆ deficiency would not be expected to cause elevations ofMMA. Thus, again it is not clear which vitamin, or pair of vitamins, orwhether all three vitamins is or are deficient. One could pick a singlevitamin with which to treat a patient for several weeks or months, andthen repeat measurement of metabolite levels to determine if a partialor full correction had occurred. If there was no response, one could tryanother vitamin, or if there was a partial response one could add asecond vitamin, and then repeat metabolite measurement after severalweeks or months. If there was still no response, one could try the thirdvitamin, or if there was a partial response, one could try a differentpair of vitamins. Eventually one could determine whether an individualvitamin, a particular pair of vitamins, or all three vitamins wererequired to normalize or maximally reduce the metabolite levels, but itwould often require months or even a year to make this determination.Such a determination would be expensive. In addition, a patient who wasoptimally treated with a single vitamin or pair of vitamins mightsubsequently develop a deficiency of one or even two of the othervitamins as evidenced by a re-elevation or increase in the levels of oneor more metabolites. Therapeutic testing could be reinitiated andcontinued as described above, although this would also be time-consumingand expensive.

[0069] It requires less time and expense to treat patients with elevatedmetabolite levels with a combination of vitamin B₁₂ and folate, or acombination of vitamin B_(12,) folate and vitamin B₆. The utility of theapproach of the present invention is appreciated only after it istaught, for the first time in the present disclosure, that a deficiencyof one or more of the three vitamins occurs commonly in the elderlypopulation as evidenced by elevation of one or more metabolites, i.e.,MMA, 2-MCA, HC and CT. TABLE 2 SERUM METABOLITE & VITAMIN LEVELS IN AGERIATRIC OUTPATIENT POPULATION Total Patient B₁₂ Folate HomocysteineMMA CT MC 116 66** 9.8 41.8** 1508** 507** 759** 118 79** 9.3 29.6**2200** 343** 428** 016 155** 7.6 15.3 1316** 208 196 067 163** 6.6 9.993 164 69 091 178** 12.0 29.2** 3108** 438** 318** 042 181** 11.3 13.0452** 300 262** 030 185** 6.6 26.0** 282** 310 223 037 187** 9.4 12.3160 218 334** 100 187** 9.5 13.6 208 453** 141 036 188* 9.9 16.3** 298**385** 322** 109 189** 7.6 12.3 127 188 161 007 191** 11.7 67.1** 6349**619** 1005** 018 193** 5.8 16.7** 412** 272 235** 050 210 4.0 25.3**464** 727** 121 108 214 6.0 31.1** 264 523** 315** 041 216 7.2 19.1**418** 360** 288* 126 224 6.5 8.8 103 361** 121 005 231 12.5 17.1** 269825** 276** 024 235 13.0 18.5** 2946** 232 289** 111 237 6.3 14.6 135380** 203 023 239 4.1 21.9** 385** 775** 279** 010 256 12.9 11.5 652**119 144 055 258 6.8 7.5 189 342 185 102 259 10.9 23.9** 1894** 423**400** 026 260 18.5 20.4** 1949** 295 248** 107 262 13.1 10.1 231 628**153 038 269 7.6 15.7 222 152 152 140 277 4.0 29.1** 744** 602** 254**074 278 5.2 24.1** 699** 296 187 002 278 14.6 14.8 554** 259 277** 019282 8.5 12.4 329** 262 161 035 287 5.8 9.8 230 390** 218 049 290 3.933.0** 140 275 138 078 290 10.9 12.5 197 240 209 045 291 8.7 9.5 162613** 132 092 294 14.9 19.3** 500** 246 167 137 297 6.8 10.1 631** 340184 072 298 6.7 19.7** 375** 302 246** 149 310 8.3 16.1 314** 199 149047 312 4.9 15.9 277** 271 173 060 312 9.4 8.0 100 228 203 046 314 6.516.2 142 336 125 093 318 6.4 16.5** 304** 361** 130 014 321 14.5 10.7275** 233 170 088 327 7.1 17.8** 263 507** 258** 032 340 6.6 8.6 150 133133 147 347 7.6 18.2** 305** 219 265** 001 351 4.7 20.8** 199 402** 223090 353 4.9 20.7** 144 419** 178 008 358 5.4 11.6 372** 529** 177 104360 12.7 12.1 260 89 77 110 370 3.0** 17.1** 456** 297 150 103 371 18.714.5 257 219 180 056 373 6.5 12.4 236 415** 189 048 374 3.6** 9.7 167237 230** 131 377 10.9 13.6 256 220 85 122 378 76 21.9** 906** 227 196004 385 8.6 10.3 109 288 92 120 390 9.8 22.9** 499** 529** 260** 138 4056.9 14.7 334** 238 188 141 407 8.1 14.3 168 259 263** 101 408 5.9 9.2160 134 40 145 410 3.7** 25.4** 567** 550 349** 027 415 11.1 10.6 169278 164 028 418 5.6 34.6** 608** 589** 351** 011 420 10.6 18.8** 683**1014** 282** 081 421 6.6 16.5** 861** 641** 531** 033 423 4.2 16.3** 156194 170 057 425 18.3 13.5 209 381** 321** 021 427 18.9 12.1 223 524**168 135 430 8.8 13.5 284** 412** 180 097 435 15.4 10.9 353** 465** 119052 438 6.8 15.2 281** 372** 238** 132 448 12.6 16.8** 1931** 394**250** 086 451 12.1 6.6 139 208 107 148 458 13.9 11.4 187 322 238** 012466 15.3 8.3 560** 250 144 083 466 12.0 13.7 366** 214 193 133 470 13.810.8 290** 275 55 017 475 4.0 39.6** 196 467** 220 053 476 13.4 12.3 226206 125 009 482 6.5 25.3** 240 470** 214 066 498 9.6 12.9 374** 233 92031 507 11.0 14.8 173 278 220 099 507 10.4 9.6 124 233 63 128 507 4.69.4 294** 324 176 013 514 11.3 15.9 163 151 522 7.8 14.3 370** 324 215077 523 6.8 17.7** 184 210 214 079 523 15.6 13.0 316** 223 251** 054 5244.9 10.0 148 230 123 020 524 9.9 14.2 235 366** 190 069 528 7.0 9.7 257281 83 085 536 4.0 22.5** 97 191 114 084 551 14.2 12.5 166 179 131 082559 12.3 14.6 208 371** 182 117 560 3.4** 18.8** 102 176 88 061 561 12.79.8 170 404** 152 006 567 4.6 16.8** 138 688** 165 129 567 4.9 16.2363** 495** 331** 003 570 11.4 12.9 189 330 230** 115 576 6.3 17.8** 128231 95 089 578 10.3 12.0 147 258 236** 143 581 2.6** 42.7** 165 555**208 114 583 5.1 16.6** 599** 660** 177 080 593 9.5 18.0** 208 289 142015 598 7.0 12.4 167 381** 95 039 598 9.6 18.1** 691** 719** 354** 070612 5.6 13.7 197 296 82 051 622 12.9 8.3 119 246 150 139 628 8.5 7.8 145166 83 150 628 8.6 14.5 295** 315 183 043 635 5.9 13.7 239 272 189 096651 17.4 9.7 326** 073 657 7.0 9.5 186 283 78 127 665 5.8 8.1 166 344**147 121 677 10.2 9.5 226 346** 173 034 694 15.9 12.1 406** 592** 584**124 697 9.7 11.0 63 179 60 123 702 10.4 10.6 186 148 96 113 705 7.6 8.4107 534** 92 071 709 10.6 11.3 207 584** 141 076 722 8.1 10.5 271 489**138 044 724 7.3 12.1 212 683** 217 040 731 15.1 7.4 205 149 136 062 7414.4 18.7** 153 856** 416** 025 741 10.0 12.2 224 344** 121 119 755 5.910.1 187 377** 61 075 757 10.0 24.7** 246 345** 276** 098 759 13.8 13.9380** 239 156 134 769 7.5 10.4 125 131 81 087 773 25.0 10.1 181 285 135142 788 4.6 12.1 166 273 129 064 792 15.4 8.6 218 299 139 094 793 16.610.0 186 179 173 022 808 8.8 14.4 184 271 161 112 812 12.0 9.2 181 184108 125 817 14.4 11.0 158 242 72 106 862 5.3 9.2 94 300 95 146 890 13.911.9 135 058 897 5.3 18.5** 154 460** 80 063 943 17.8 19.7** 277** 642**306** 095 960 25.3 10.7 135 181 111 152 963 9.4 8.8 198 130 971 15.913.5 106 307 84 059 1063 9.4 9.7 129 378** 54 105 1109 11.0 6.1 87 15564 136 1163 6.0 13.1 250 565** 122 065 1251 14.5 10.7 88 147 88 029 149022.2 9.7 129 111 105 144 1536 7.0 17.7** 216 694** 418** 068 1809 12.710.4 59 128 39

[0070] TABLE 3 SERUM METABOLITE & VITAMIN LEVELS IN A GERIATRIC NURSINGHOME POPULATION Patient B₁₂ Folate Homocysteine Methylmalonic Acid NH170  8** 14.0 34.8**  3365** NH129  40** 7.4 40.9**  6245** NH156  44**22.4 17.4**  1130** NH139  56** 9.7 20.9**  1180** NH132  67** 7.692.4** 12641** NH176  129** 9.2 20.3**  1156** NH196  136** 6.2 41.0** 1077** NH109  139** 9.8 20.9**  1294** NH203  146** 4.3 12.2  437**NH141  161** 13.4 12.2  223 NH178  172** 8.2 5.9  141 NH103  189** 5.513.1  362** NH181  196** 6.3 14.7  296** NH160  206 11.9 12.5  640**NH197  221 24.0 10.5  654** NH073  222 3.6** 19.8**  490** NH110  2275.5 13.7  1297** NH010  228 4.0 21.1**  413** NH012  234 8.7 16.0  596**NH037  236 11.5 22.5** 11299** NH114  238 12.8 13.2  442** NH211  2406.0 14.1  166 NH075  250 9.3 12.1  170 NH172  255 7.2 14.4  552** NH148 259 5.7 19.2**  317** NH138  264 9.2 16.7**  340** NH150  264 4.0 13.7  98 NH099  272 5.5 12.5  125 NH124  275 6.9 11.5   87 NH179  301 7.67.1  143 NH135  302 6.5 23.4**  397** NH087  304 7.8 10.8  327** NH180 304 5.8 10.5  237 NH209  306 7.6 11.9  105 NH107  310 3.3** 8.6  148NH081  320 4.3 23.6**  470** NH068  324 7.9 13.4  243 NH183  325 7.711.1  144 NH033  330 13.8 7.7  149 NH161  333 8.5 11.3  385** NH192  33710.7 9.5  209 NH136  340 6.7 18.2**  409** NH191  342 20.2 13.4  271NH137  343 4.0 15.6  183 NH182  346 8.2 14.4  448** NH020  347 8.4 10.4 149 NH165  351 18.5 11.8  425** NH095  352 8.5 14.5  366** NH194  3614.3 20.3**  305** NH106  362 4.8 12.9  298** NH060  367 4.7 16.4**   71NH009  368 5.1 15.9  325** NH071  382 4.9 12.9  330** NH080  390 6.115.0  171 NH013  407 6.7 12.4  310** NH126  409 9.2 17.4**  137 NH030 411 11.2 10.4  844** NH210  413 8.6 11.9  210 NH158  414 5.7 16.2 508** NH027  416 10.2 15.5  769** NH003  424 16.5 9.5  167 NH187  4294.7 8.8  439** NH022  430 10.5 14.0  214 NH082  436 10.6 17.7**  340**NH162  438 6.1 19.2**  180 NH021  439 5.3 15.1  191 NH056  447 11.7 10.9 184 NH119  448 3.2** 14.1  241 NH120  448 5.6 12.0  138 NH186  450 4.723.1**  213 NH064  451 6.9 10.6  237 NH057  453 14.6 10.4  282** NH131 454 8.1 16.2  258 NH059  462 6.0 9.1  147 NH202  465 3.3** 17.0** 393** NH134  475 15.3 11.6  321** NH083  475 7.4 10.6  178 NH199  47915.1 10.4  141 NH042  482 6.0 15.0  141 NH200  491 13.6 9.8  154 NH213 497 8.1 10.0   92 NH143  500 5.2 22.1**  175 NH031  502 6.4 16.1  151NH188  504 12.5 15.1  1461** NH171  504 10.7 12.9  344** NH008  505 4.69.9  185 NH102  506 16.6 9.1  236 NH145  512 7.7 22.2**  161 NH093  5145.1 17.7**  185 NH118  524 25.0 10.1  314** NH185  524 8.7 12.1   84NH111  527 5.1 18.4**  250 NH149  530 12.6 18.2**  531** NH011  534 8.112.5  654** NH128  540 4.3 11.6  120 NH035  547 7.5 9.8  193 NH005  55117.7 5.0  365** NH212  552 11.9 12.1  202 NH007  554 6.4 26.1**  646**NH086  554 9.5 5.1  127 NH069  555 22.7 6.8  134 NH121  555 8.2 10.0 112 NH117  571 6.6 9.7  351** NH055  581 14.8 9.1  265 NH025  581 5.215.3  181 NH104  583 3.9 14.6  1699** NH173  583 11.2 10.6  160 NH177 584 6.2 5.7  111 NH207  586 8.5 16.4**  243 NH070  591 5.4 12.0  168NH038  592 8.0 8.8  230 NH049  599 10.7 21.7**  238 NH062  606 4.5 7.7  96 NH153  608 7.7 13.6  221 NH206  611 6.6 16.4**  400** NH018  6146.3 10.9  123 NH163  616 5.0 9.6  132 NH189  619 7.6 12.0  158 NH045 620 21.0 12.4  265 NH074  621 10.2 9.2  172 NH054  623 8.0 9.8  121NH152  625 8.2 7.8  206 NH140  637 21.7 13.6  300** NH050  642 16.3 13.5 275** NH089  644 7.7 16.7**  444** NH036  649 7.9 10.7   68 NH097  6516.6 13.4  426** NH016  656 4.1 61.0**  356** NH053  657 14.2 10.6  320**NH066  658 7.7 11.4  228 NH051  659 4.0 10.7  216 NH108  671 5.8 24.0** 823** NH058  673 6.0 11.2  392** NH028  675 22.3 9.1  105 NH204  6784.7 10.2  148 NH169  679 6.9 19.2**  267 NH032  681 12.7 5.9   99 NH065 682 11.0 13.5  176 NH061  683 13.4 9.6  190 NH116  685 9.0 7.5  244NH015  699 6.8 16.8**  236 NH157  711 10.0 12.8  198 NH155  715 10.017.6**  308** NH034  715 7.9 11.4  179 NH040  717 10.5 15.7  256 NH105 718 6.0 13.2  308** NH048  719 8.0 10.8  207 NH084  720 6.8 9.4  169NH115  724 16.3 9.4  161 NH205  734 8.5 13.3  232 NH113  738 11.7 10.3 171 NH154  738 13.7 9.6  123 NH167  741 17.0 6.6  129 NH190  752 5.214.1  254 NH067  760 22.5 9.5  232 NH014  767 8.9 7.3  100 NH072  7688.3 6.9  131 NH133  772 8.8 20.4**  219 NH122  778 6.0 10.4  108 NH076 781 12.1 14.9  282** NH147  785 7.5 24.5**  411** NH026  786 9.7 8.3 146 NH151  789 24.4 11.1  182 NH198  797 10.9 10.7  158 NH088  801 6.418.3**  184 NH004  806 11.3 8.8   96 NH024  818 5.1 14.1  219 NH100  82616.4 10.5  103 NH078  831 7.2 10.3  266 NH052  844 19.6 8.0  193 NH142 848 18.6 12.1  398** NH002  862 9.4 11.3  212 NH091  891 4.9 12.6  169NH127  897 22.0 8.4  132 NH096  901 9.3 5.2  104 NH201  910 25.0 15.7 424** NH184  941 21.5 10.8  170 NH208  945 20.2 9.8  111 NH130  96822.4 10.4  339** NH164  989 8.0 16.8**  102 NH077 1006 15.1 9.2  188NH017 1015 11.9 9.5  175 NH029 1053 18.6 11.4  161 NH023 1055 9.3 9.7 193 NH047 1079 6.4 11.4  106 NH043 1082 14.5 13.9  144 NH195 1088 36.912.2  150 NH193 1092 8.2 15.7  225 NH046 1093 9.2 18.8**  186 NH101 11083.9 8.1  139 NH098 1117 11.3 12.5   88 NH168 1124 25.2 15.0  203 NH0061126 6.9 8.1  159 NH144 1135 8.0 21.9**  262 NH044 1159 26.8 10.2  109NH175 1162 7.8 12.0  210 NH146 1179 9.8 10.1  129 NH112 1238 10.3 15.0 347** NH001 1304 13.1 6.9  142 NH166 1337 13.4 8.3   67 NH079 1346 18.012.0  248 NH041 1528 20.7 8.2  155 NH063 1559 15.0 7.0   66 NH159 15666.6 15.5  451** NH125 1703 8.2 20.6**  153 NH094 1768 15.9 8.4  182NH123 2028 10.2 16.8**  206 NH174 2106 13.3 12.8  280** NH039 2227 23.88.9  119 NH019 2297 11.1 15.5  177 NH092 2360 5.7 9.8  131 NH085 314122.0 26.9**  1947**

[0071] TABLE 4 SERUM METABOLITE & VITAMIN LEVELS IN A GERIATRICPOPULATION Patient B₁₂ Folate Homocysteine MMA 495  77** 10.0 65.4**3145** 484  84** 10.0 77.5** 6820** 522  100** 3.6** 15.5  967** 455 115** 1.9** 21.8**  170 493  135** 4.4 16.9**  421** 528  145** 3.938.3**  729** 510  155** 4.6 14.1  804** 502  155** 2.1** 16.9**  347**412  160** 18.5** 33.8** 1301** 409  160** 4.8 16.8**  164 470  165**9.2 19.9** 1468** 460  165** 6.8 11.5  142 437  170** 4.9 16.5**  813439  170** 1.2** 21.3**  502** 525  175** 11.5 15.3 1058** 442  175**4.2 17.5**  328** 456  180** 7.3 11.1  206 450  180** 5.0 11.8  196 477 185** 3.4** 31.4**  369** 508  190** 4.1 19.5**  335** 423  190** 2.5**19.0**  329** 462  190** 3.8 11.6  276** 523  190** 5.6 16.8**  207 482 190** 2.9** 25.1**  179 459  190** 5.3 19.6**  167 543  195** 4.3 13.5 470** 520  195** 1.7** 22.2**  309** 431  195** 7.2 13.5  251 513  2005.0 25.0** 1184** 534  200 4.9 32.6** 1080** 515  200 4.9 17.3**  478**531  200 5.1 26.8**  466** 516  200 3.6** 17.8**  279** 526  200 1.6**23.5**  171 471  205 5.7 22.0**  542** 413  205 2.6** 20.4**  304** 497 205 3.3** 19.4**  258 539  205 4.1 15.4  247 544  205 12.5 11.7  233540  205 4.0 17.1**  185 517  205 2.2** 15.0  151 496  210 3.7** 15.21103** 488  210 16.5 21.8**  600** 416  215 12.5 10.0  197 434  220 7.124.8**  439** 545  220 11.5 14.4  407** 547  220 5.3 17.5**  396** 408 220 3.2** 16.4**  357** 449  220 3.7** 13.7  272** 507  220 8.5 10.0 179 458  225 10.5 21.1**  964** 491  225 7.2 16.0  472** 529  230 2.0**61.1 1172** 415  230 3.2** 28.9**  377** 453  230 3.6** 19.8**  336**448  230 5.2 13.1  319** 498  230 5.9 20.1**  255 533  230 5.7 11.7  151466  235 35.0 12.1  617** 537  235 5.7 10.7  394** 483  235 8.6 16.6** 344** 512  235 3.9 12.5  190 452  240 4.7 26.5** 1068** 454  240 5.211.9  201 535  240 4.4 15.3  195 421  245 10.5 12.5  464** 469  245 6.220.0**  448** 474  245 7.3 10.3  327** 486  245 9.2 12.6  156 536  25022.5 20.3** 1068** 475  250 5.6 23.0  456** 511  250 2.7** 23.1**  398**465  250 4.1 23.1**  323** 506  250 5.2 11.5  252 417  250 5.5 25.2** 241 524 1250 2.5** 14.4  212 411  250 9.9 11.5  200 492  250 5.2 10.7 182 548  250 2.9** 12.4  179 441  250 4.5 8.5  147 480  255 4.8 16.9** 558** 532  255 7.0 14.8  419** 464  255 11.5 12.9  400** 494  255 6.212.1  293** 106  255 4.5 11.7  203 546  260 5.5 14.7  662** 541  260 5.430.8**  426** 420  260 5.3 13.6  347** 500  260 6.7 14.0  330** 538  2609.3 17.3**  298** 457  260 2.9** 12.6  286** 472  260 8.3 13.8  278**424  260 8.3 10.1  242 433  260 6.8 10.5  197 425  265 7.3 14.7  724**468  265 3.8 16.7**  289** 435  265 7.4 14.0  150 499  265 2.2** 12.4 131 432  270 4.3 28.3**  432** 521  270 3.7** 15.3  349** 549  270 4.2112.4  343** 518  270 10.0 10.1  276** 418  270 26.0 9.4  213 419  2706.5 12.5  212 428  270 4.2 18.7**  189 443  270 8.8 12.0  187 446  27011.0 8.1  157 461  275 7.6 15.1  663** 440  275 4.9 12.9  248 436  2756.3 30.1**  233 530  275 7.4 13.6  231 438  275 4.6 8.5  221 527  2757.5 10.5  219 444  275 4.0 12.2  180 429  280 5.3 15.3  463** 503  2804.4 25.7**  421** 485  280 3.5** 15.6  381** 410  280 14.5 10.0  201 487 280 3.9 10.5  166 430  280 9.2 8.8  161 519  285 3.9 22.2**  919** 476 285 10.5 12.8  339** 509  285 5.4 13.0  331** 501  285 5.5 12.4  252542  285 6.9 15.5  242 445  285 7.2 14.9  237 427  285 4.0 17.1**  233490  290 4.7 13.9  203 451  290 2.1** 20.0**  226 414  290 7.0 9.7  117467  290 4.1 6.5  68 463  295 5.8 12.3  296** 473  295 7.5 14.4  290**505  295 4.1 12.4  257 198  300 11.5 10.9  323** 195  300 9.8 12.2  216207  305 7.7 13.2  330**  67  305 8.6 15.4  312**  50  305 9.0 11.6  235 70  305 12.5 12.7  228 113  305 5.6 13.5  201  39  305 6.9 19.7**  170 3  305 4.2 11.5  135 325  305 14.5 9.4  94 368  310 4.7 15.9  371** 322 310 7.8 15.3  362** 295  310 7.2 13.8  305** 347  310 5.8 16.5**  266313  310 6.1 16.5**  219 355  310 5.5 15.4  138 291  310 4.5 15.2  125478  315 23.0 17.7**  857**  53  315 5.8 12.1  505** 240  315 6.7 12.3 394**  14  315 9.6 14.2  331** 137  315 7.8 24.3**  306** 254  315 8.717.0**  285** 109  315 3.7** 16.5**  263 252  315 5.2 10.1  241 186  3154.1 15.4  238 183  315 5.5 10.7  195 390  315 6.9 10.0  188 267  3152.2** 12.0  124 310  320 12.0 13.8  395**  31  320 17.0 12.9  334**  88 320 4.8 13.8  217 403  320 9.6 11.3  162  60  320 6.2 11.4  155 315 320 6.4 9.9  136 175  325 6.3 17.8**  486** 317  325 22.0 14.0  294** 18  325 6.3 11.1  241 247  325 13.5 13.2  231 223  325 9.2 12.6  203132  325 3.7** 15.4  184 168  325 4.3 10.2  174 238  325 5.5 9.9  166117  325 5.2 15.0  154 404  330 2.5** 33.1** 1085** 138  330 4.8 11.3 360** 316  330 3.6** 10.2  272**  61  330 5.1 12.5  242 333  330 34.09.2  235  16  330 4.6 13.3  211 276  330 5.7 11.9  200 391  330 4.1 8.4 184 362  330 9.2 11.7  178  1  330 9.9 8.9  170 379  335 16.0 12.1 471** 147  335 9.0 9.7  427**  89  335 8.0 15.3  385** 211  335 5.012.2  374**  45  335 5.9 16.3**  250  47  335 5.0 13.6  249 402  335 4.713.5  230 314  335 7.6 9.7  203 150  335 4.8 11.2  119 120  340 1.9**21.0**  775** 284  340 7.2 25.6**  439** 230  340 14.0 11.4  419** 149 340 8.8 18.9**  337** 269  340 3.9 16.2  302** 197  340 10.5 12.8  233 19  340 9.6 11.0  232 422  340 3.1** 14.4  188 196  340 11.5 8.9  169 40  345 8.7 14.6  610** 244  345 8.6 15.8  461** 287  345 5.7 18.1** 427** 100  345 8.3 14.8  403** 383  345 4.3 27.2**  284**  62  345 19.59.6  250 350  345 8.0 10.0  249  65  345 8.0 10.2  247 307  345 16.511.6  208  69  345 17.0 9.9  197 328  345 7.5 8.9  192  43  345 6.0 13.2 191 222  345 6.1 9.2  175 306  345 4.3 17.2**  160 154  345 7.1 10.2 148  94  350 4.8 16.1  302** 201  350 6.1 9.9  200  13  350 5.1 10.9 193 236  355 7.2 14.8  309** 191  355 5.8 15.3  257 481  355 5.2 17.1** 134  92  360 4.2 25.2**  321** 324  360 3.8 16.6**  264  87  360 3.3**13.3  200  46  360 5.4 11.1  179 289  360 9.5 7.9  129 392  360 5.1 10.3 125 320  365 6.4 17.3**  240 134  365 13.5 11.8  238 239  365 7.7 13.2 236 326  365 6.0 10.9  180 364  365 4.1 13.9  154 218  365 7.5 11.2 126 216  365 6.2 12.2  119 248  365 5.7 13.3  117 375  370 4.1 20.7** 532** 288  370 6.4 18.8**  436** 161  370 6.3 11.2  340** 244  370 19.59.8  286** 330  370 18.0 12.2  228 334  370 12.5 8.7  172 275  370 6.912.7  162  54  375 7.3 10.1  583** 185  375 9.3 10.5  386**  52  375 8.115.5  291** 366  375 5.0 12.5  280**  93  375 3.3** 16.2  248 151  3752.9** 12.3  235  85  375 6.7 14.8  217 294  375 7.0 12.2  184 361  3757.9 10.7  179 318  375 5.5 13.7  160 386  375 7.6 10.4  153 304  375 9.19.4  132 228  380 7.7 17.1**  320** 110  380 4.0 7.2  135 204  380 5.710.6  91 348  385 2.3** 17.4**  368** 146  385 11.5 12.5  253 260  3855.5 13.7  211 136  385 3.6** 19.8**  205 338  385 5.0 16.2  180 376  3853.6** 13.7  154 194  385 12.5 7.9  153 504  385 38.0 9.5  138 160  3908.1 24.7**  475** 354  390 11.5 12.8  212  25  390 5.1 11.3  205 387 390 8.7 8.4  162  86  390 21.0 12.6  133 133  390 3.9 11.3  113 331 395 12.0 20.1**  638** 130  395 10.5 10.8  256  82  395 2.8** 9.8  236119  395 12.5 16.3**  209 380  395 10.5 14.3  159 373  395 5.5 11.6  152256  395 10.5 9.9  149 384  395 7.3 14.7  116 105  400 19.0 10.5  322**251  400 4.8 14.9  289** 352  400 11.5 9.6  181 279  400 4.5 11.7  170339  400 7.4 13.6  168 381  405 6.7 12.4  294** 285  405 7.0 14.2  281**340  405 3.6** 19.6**  275**  51  405 6.5 14.3  233  33  405 6.5 9.6 207 268  405 3.3** 14.9  205  73  405 5.2 13.1  172  17  410 7.5 16.2 473** 286  410 4.7 18.8**  415** 140  410 5.9 21.7**  302** 116  4106.8 14.5  218 396  410 5.6 16.1  190 356  410 1.9** 27.6**  149 237  4103.6** 16.6**  122 112  410 5.5 8.9  107 259  410 4.7 11.6  99 176  4155.2 21.9**  453** 193  415 10.5 11.3  163 323  415 6.1 9.6  163 202  41511.5 9.4  150 398  415 8.0 12.6  134 321  420 5.2 10.7  383** 142  42029.0 8.3  234 327  420 3.2** 14.6  203 342  420 7.3 9.4  156 170  42020.5 10.3  142 345  420 29.5 13.2  136 302  420 8.6 8.8  128 115  4256.3 22.2**  628**  97  425 12.5 19.8**  313** 246  425 8.7 15.1  241  72 425 10.5 13.5  241 365  425 6.7 16.7**  237 139  425 12.5 10.4  224 143 425 8.1 13.5  216 426  425 19.5 14.5  201 303  425 3.0** 14.5  154 388 425 6.2 12.3  135 127  425 6.7 8.4  100 262  430 10.0 12.1  323** 270 430 4.8 12.9  293** 514  430 4.3 12.9  197 341  430 3.5** 19.9**  190278  430 5.2 10.8  182 370  430 11.0 15.3  174  55  430 7.6 11.0  162274  430 5.0 8.2  131 367  430 17.5 8.0  126  98  430 13.5 12.8  125 337 435 13.5 14.1  395** 309  435 8.7 12.9  349** 305  435 17.5 15.4  187144  435 25.0 8.9  167  34  435 8.6 7.6  157 234  435 9.7 9.2  116 123 440 9.6 12.2  622** 200  440 4.8 12.4  257 250  440 7.5 12.9  248 107 440 6.3 14.7  183 300  440 6.5 7.9  123 374  445 5.4 14.0  247 372  44511.0 11.0  181  36  445 4.0 10.0  181 271  445 7.2 10.4  124 242  44515.5 9.6  112 264  445 6.0 10.7  100 172  450 11.5 14.9  607**  32  45011.5 13.6  362** 346  450 13.5 15.8  330**  41  450 8.5 11.4  194  95 450 5.1 12.5  182 357  455 6.3 14.4  296** 319  455 17.0 10.2  147 308 455 15.0 9.8  131 235  455 23.0 9.0  114 349  455 9.2 8.3  82 178  4605.6 20.6**  473** 312  460 4.7 14.4  197  79  460 5.0 10.4  173 131  46018.0 10.2  162 243  460 2.6** 11.6  160 261  465 7.7 10.6  252 378  4655.4 13.2  221  49  465 47.0 10.8  179 226  465 7.7 10.2  173 377  4655.6 8.5  143 253  465 10.0 7.0  138  76  470 12.5 14.8  304** 203  47015.0 7.6  233 296  470 23.5 11.0  161 382  470 5.3 11.1  109  6  47510.5 12.5  232  75  475 4.5 8.1  150 332  475 9.4 10.0  144 290  47514.0 9.1  143 128  475 5.9 9.3  133 124  475 6.0 13.5  111 177  475 8.89.1  106 126  480 11.0 11.0  212 283  480 5.2 10.6  175 209  480 10.510.5  175 293  480 6.8 15.5  135 121  485 4.7 20.0**  345** 282  48512.0 10.9  236  71  485 13.5 8.1  168 385  485 9.0 14.1  128 190  4959.9 10.4  410** 210  495 8.6 12.0  243 155  495 5.9 10.4  219 336  49513.5 9.9  135 280  500 8.7 14.5  334**  96  500 4.7 10.8  237 145  5005.9 17.5**  233 199  500 4.2 13.8  199 489  500 11.5 9.7  198 217  5006.4 9.6  166  90  500 7.5 8.5  106 164  510 5.2 23.8**  408** 343  5104.5 13.7  284**  42  510 4.9 7.4  233 351  510 8.5 11.0  207 299  51012.0 8.0  104  99  520 10.5 25.8**  322** 114  520 30.0 10.9  220 369 520 29.0 16.7**  206  37  520 10.5 8.6  191 215  520 6.7 16.8**  151401  520 7.5 12.6  148 229  520 7.9 11.0  116 135  520 3.2** 8.3  88  81 530 6.8 14.8  372**  91  530 14.5 10.6  228 167  530 23.5 9.2  176 181 530 5.5 9.3  171  56  530 20.0 8.3  163  5  530 13.5 8.1  159 180  54012.0 9.0  216 311  540 4.1 13.3  214 389  540 3.9 13.9  169 125  540 5.513.0  159  35  540 22.5 11.0  123 104  550 10.5 16.5**  544** 393  5504.9 11.9  339** 394  550 23.0 14.0  278** 292  550 6.9 16.2  263 163 550 6.7 14.3  219  66  550 10.5 11.6  206  29  550 17.5 9.6  191 227 550 7.9 11.7  154  38  550 7.5 11.9  152 241  550 10.5 9.8  100 102 550 9.7 8.6  91  77  560 24.0 14.8  554** 162  560 10.5 11.8  275** 273 560 8.7 9.4  180  80  560 6.3 11.2  108 255  560 8.8 9.9  93 122  57066.0 13.8  304** 208  570 34.0 10.2  255  23  570 21.5 8.3  241 447  57025.0 10.0  164 225  570 5.7 12.2  154 174  570 7.1 11.0  127  11  57019.0 8.9  113 165  580 10.5 14.8  226 182  580 8.9 8.2  189 245  59015.5 10.0  262  83  590 17.5 8.3  199 166  590 11.5 9.4  188 158  5907.3 10.7  166 187  590 4.5 11.0  146 156  590 23.5 11.3  112 231  6009.5 9.0  192  78  600 11.5 9.4  151 329  610 15.0 7.3  312**  57  61016.0 11.9  286**  7  610 12.0 10.4  195 277  610 9.5 7.8  153 108  62013.5 8.4  191 205  620 18.0 7.5  145 263  620 9.8 10.2  101  9  630 4.911.4  300** 111  630 8.3 11.1  276**  68  630 11.5 8.9  143 399  63014.0 11.0  90 266  640 5.1 15.7  364**  12  640 24.5 9.0  233 152  6408.1 10.0  209 405  640 7.0 12.8  186  27  640 22.5 8.4  136 258  640 8.311.2  120 249  640 8.7 9.1  81 297  650 16.0 10.0  279** 192  650 4.914.9  213 257  650 3.3** 16.3**  208 184  650 12.5 9.9  193  58  65018.5 10.7  172 301  650 16.0 15.5  162 397  650 12.5 8.4  146 272  65011.0 7.4  120 153  650 7.1 13.1  116 406  650 6.6 5.8  81  10  660 9.07.6  154  26  660 22.0 8.3  132 265  670 3.9 19.3**  509** 359  670 21.08.3  269  48  670 32.0 9.9  262 335  670 11.5 8.1  121 189  680 6.617.9**  358** 220  680 15.5 10.9  115  15  690 13.5 13.4  159  44  70020.0 12.7  244  21  700 13.5 10.2  129  74  700 15.0 7.1  65  4  71029.0 8.5  266 353  710 11.5 11.4  206 281  710 10.5 9.6  185  2  710 8.08.5  109 212  740 20.0 11.1  250  8  740 12.0 11.5  216 206  750 12.58.3  116 101  770 14.5 12.7  372** 344  770 32.0 11.7  297**  20  77035.0 10.1  245 407  770 10.5 12.0  110 360  780 2.7** 20.9**  157 232 790 15.5 10.1  151 141  790 12.5 9.5  74 129  800 8.7 11.7  211 188 800 15.0 12.3  174 400  800 12.5 10.3  156  24  810 23.0 7.5  194 173 830 35.0 11.4  243 214  830 21.5 12.0  187  63  830 13.8 8.8  185 148 830 45.0 7.1  146  84  830 23.5 7.0  136 179  830 16.5 6.6  96 171  84023.5 11.2  195  28  870 5.8 15.9  197 233  870 7.9 12.7  169 221  87040.0 7.0  126 371  880 20.0 8.5  152 213  890 10.5 18.0**  231 358  90021.0 8.3  149 298  910 15.5 10.2  221 118  910 100.0 9.7  170 479  95011.5 12.1  188  30  950 6.2 10.5  170 159 1000 9.5 8.7  281** 219 105037.0 14.3  313** 103 1050 12.5 10.3  154  59 1150 17.5 7.3  180 157 125012.0 14.0  206 363 1350 28.0 10.4  190  22 1400 13.5 10.4  233  64 140031.0 9.7  149 169 1450 15.0 9.5  150

What is claimed is:
 1. A method of preventing vascular disease in ahuman, comprising periodically administering orally a single formulationhaving between 0.3 and 10 mg vitamin B₁₂ and 0.1 and 0.4 mg folic acid.2. The method of claim 1 wherein the formulation includes approximately2 mg vitamin B₁₂ and 0.4 mg folic acid.
 3. The method of claim 1 whereinsaid vascular disease is cardiovascular disease.
 4. The method of claim1 wherein said vascular disease is stroke.
 5. The method of claim 1wherein said vascular disease is peripheral vascular disease.
 6. Themethod of claim 1 wherein said vascular disease is associated with renaldisease.
 7. A method of preventing vascular disease in a human,comprising periodically administering orally a single formulation havingbetween 0.3 and 10 mg vitamin B_(12,) and 0.1 and 0.4 mg folic acid, and5 and 75 mg vitamin B6.
 8. The method of claim 7 wherein the formulationincludes approximately 2 mg vitamin B12 and 0.4 mg folic acid and 25 mgvitamin B6.
 9. The method of claim 7 wherein said vascular disease iscardiovascular disease.
 10. The method of claim 7 wherein said vasculardisease is stroke.
 11. The method of claim 7 wherein said vasculardisease is peripheral vascular disease.
 12. The method of claim 7wherein said vascular disease is associated with renal disease.
 13. Amethod of preventing vascular disease in a human, comprisingperiodically administering orally a single formulation having between0.3 and 10 mg vitamin B12 and 0.4 and 10.0 mg folic acid.
 14. The methodof claim 13 wherein the formulation includes approximately 2 mg vitaminB12 and 1.0 mg folic acid.
 15. The method of claim 13 wherein theformulation includes approximately 2 mg vitamin B12 and 2.0 mg folicacid.
 16. The method of claim 13 wherein the formulation includesapproximately 2 mg vitamin B12 and 2.5 mg folic acid.
 17. The method ofclaim 13 wherein the formulation includes approximately 1 mg vitamin B12and 2.0 mg folic acid.
 18. The method of claim 13 wherein theformulation includes approximately 1 mg vitamin B12 and 2.5 mg folicacid.
 19. The method of claim 13 wherein said vascular disease iscardiovascular disease.
 20. The method of claim 13 wherein said vasculardisease is stroke.
 21. The method of claim 13 wherein said vasculardisease is peripheral vascular disease.
 22. The method of claim 13wherein said vascular disease is associated with renal disease.
 23. Amethod of preventing vascular disease in a human, comprisingperiodically administering orally a single formulation having between0.3 and 10 mg vitamin B12, and 0.4 and 10 mg folic acid, and 5 and 75 mgvitamin B6.
 24. The method of claim 23 wherein the formulation includesapproximately 2 mg vitamin B12 and 1.0 mg folic acid and 25 mg vitaminB6.
 25. The method of claim 23 wherein the formulation includesapproximately 2 mg vitamin B12 and 2.0 mg folic acid and 25 mg vitaminB6.
 26. The method of claim 23 wherein the formulation includesapproximately 2 mg vitamin B12 and 2.5 mg folic acid and 25 mg vitaminB6.
 27. The method of claim 23 wherein the formulation includesapproximately 1 mg B12 and 2.0 mg folic acid and 25 mg vitamin B6. 28.The method of claim 23 wherein the formulation includes approximately 1mg B12 and 2.5 mg folic acid and 25 mg vitamin B6.
 29. The method ofclaim 23 wherein said vascular disease is cardiovascular disease. 30.The method of claim 23 wherein said vascular disease is stroke.
 31. Themethod of claim 23 wherein said vascular disease is peripheral vasculardisease.
 32. The method of claim 23 wherein said vascular disease isassociated with renal disease.